The overall goal of this proposal is to understand the structural and molecular basis for the mechanisms of the microsomal monooxygenation system that is responsible for the oxidative metabolism of various xenobiotics and endogenous compounds. The two key components of this system are cytochromes P450 (P450s) and NADPH-cytochrome P450 oxidoreductase (CYPOR). Although we know much about the mechanisms and structures of each of these two partners individually, their interactions with each other are poorly understood. In humans, the single CYPOR protein is capable of interacting with ~50 microsomal P450s, as well as the non-P450 enzymes, heme oxygenases (HO-1 and HO-2). Furthermore, mutations of the POR gene, encoding CYPOR, result in a wide range of phenotypes, from embryonic lethality, Antley-Bixler Syndrome (ABS) to mild steroidogenic disorders. In contrast to the previously obtained closed form structure of CYPOR, our recently obtained variant form of CYPOR (CYPORTGEE) adopts an open conformation that is capable of reducing P450s, and forms stable complexes with its partners. We will use CYPORTGEE in our EPR and crystallization studies as outlined in the following specific aims. Aim 1: To determine conformational changes of CYPOR upon binding to its electron transfer partners and the interactions between them by site-directed spin-labeling EPR methods. Using the crystal structures of wild type CYPOR and CYPORTGEE as guides, double cysteine mutants (one in each of the two flavin domains) will be spin labeled, and the spin-spin distances will be measured. Spin-labeled CYP2B4 and HO-1 will also be used with single labeled CYPOR to measure the spin- spin distances between the two partners in the complex. From the distances between the two domains of CYPOR and between the two partners, structures of the complexes of CYPOR-2B4/HO-1 will be constructed. The resulting model structures of complexes of CYPOR and its partners will be validated by biochemical characterization combined with site-specific mutagenesis. Aim 2: To determine the crystal structures of complexes of CYPOR with CYP2D6, CYP3A4, CYP2B4, and human HO-1. We will use both human and rat CYPORTGEE proteins that form stable complexes with the partners. Soluble forms of CYP2B4, CYP3A4, and CYP2D6 and the soluble form of HO-1 will be used in co-crystallization studies with CYPORTGEE. The successful completion of the above studies will impact the P450 field by providing the answer to the central question in the field: What is th basis for recognition and the mechanism of electron transfer between CYPOR and its redox partners, including P450s and HO? The results from these studies will be used for drug development and designing of effective therapies by providing a better understanding of drug metabolism and the mechanism of HO-1 functions, respectively, and possibly to the design of therapeutics for CYPOR deficiencies resulting in abnormal steroidogenesis.